author manuscript nih public access jin-wu nam kyle kai-how … · chanseok shin1,2,3,4,6, jin-wu...

Expanding the MicroRNA Targeting Code: Functional Sites withCentered Pairing

Chanseok Shin1,2,3,4,6, Jin-Wu Nam1,2,3,6, Kyle Kai-How Farh1,2,3,5,6, H. RosariaChiang1,2,3, Alena Shkumatava1,2,3, and David P. Bartel1,2,3,*1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA2Howard Hughes Medical Institute3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA4Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-921, Republic ofKorea5Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge,MA 02139, USA

SUMMARYMost metazoan microRNA target sites have perfect pairing to the seed region, located near themiRNA 5’ end. Although pairing to the 3’ region sometimes supplements seed matches orcompensates for mismatches, pairing to the central region has been known to function only at raresites that impart Agronaute-catalyzed mRNA cleavage. Here we present “centered sites,” a class ofmicroRNA target sites that lacks both perfect seed pairing and 3’-compensatory pairing and insteadhas 11–12 contiguous Watson–Crick pairs to the center of the microRNA. Although centered sitescan impart mRNA cleavage in vitro (in elevated Mg2+), in cells they repress protein output withoutconsequential Agronaute-catalyzed cleavage. Our study also identified extensively paired sites thatare cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavagetargets and the identification of the centered site type help explain why central regions of manymicroRNAs are evolutionarily conserved.

INTRODUCTIONMicroRNAs (miRNAs) are a class of ~23 nucleotide (nt) RNAs that direct the post-transcriptional repression of protein-coding genes (Bartel, 2004). After processing from hairpinprecursors, miRNAs are loaded into Argonaute-containing silencing complexes, which down-regulate mRNA targets through two distinct modes, either Argonaute-catalyzed cleavage or asecond mode that involves mRNA destabilization and translational repression, at least in partthrough poly(A) shortening (Filipowicz et al., 2008).

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NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2010 December 1.

Published in final edited form as:Mol Cell. 2010 June 25; 38(6): 789–802. doi:10.1016/j.molcel.2010.06.005.

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Argonaute-catalyzed cleavage of the target strand occurs in the context of extensive basepairing, at the linkage joining the mRNA nucleotides that pair to miRNA positions 10 and 11(Elbashir et al., 2001b; Hutvagner and Zamore, 2002; Yekta et al., 2004). In mammals, thisslicing activity is catalyzed by Argonaute2 (AGO2), which leaves a 3’ hydroxyl on the 5’cleavage fragment and a 5’ monophosphate on the other (Liu et al., 2004; Meister et al.,2004; Schwarz et al., 2004). Unlike miRNAs in plants, very few examples of miRNA-dependent cleavage targets have been reported in mammals (Yekta et al., 2004; Davis et al.,2005; Jones-Rhoades et al., 2006). Nonetheless, artificially designed small interfering RNAs(siRNAs) that silence target genes through this mechanism are widely used reagents,illustrating that in principle the cleavage mode of repression can function in many contexts andfor many targets (Elbashir et al., 2001a).

Sites that confer slicing-independent destabilization and translational repression typically pairto the 5’ region of the miRNA, centering on miRNA nucleotides 2–7, known as the miRNAseed (Lewis et al., 2005; Bartel, 2009). Introducing an siRNA/miRNA or deleting anendogenous miRNA leads to modest yet detectable changes in the output of hundreds of genescontaining seed sites in their 3’UTRs (Krutzfeldt et al., 2005; Lim et al., 2005; Giraldez et al.,2006; Grimson et al., 2007; Rodriguez et al., 2007; Baek et al., 2008; Selbach et al., 2008).Moreover, most mammalian protein-coding genes are under selection to maintain pairing tothe seed of one or more miRNAs, and thousands of genes have also evolved to specificallyavoid pairing to the seeds of preferentially co-expressed miRNAs (Farh et al., 2005; Lewis etal., 2005; Stark et al., 2005; Friedman et al., 2009). These observations illustrate both the broadscope of seed-type regulation and the widespread influence of this targeting mode on mRNAevolution.

Pairing to the 3’ region of the miRNA can supplement seed pairing to enhance targetrecognition, or it can even compensate for a mismatch to the seed; such sites are known as “3’-supplementary sites” and “3’-compensatory sites”, respectively (Bartel, 2009). However,pairing to the 3’ region appears to be consequential for relatively few (<10%) of sites (Bartel,2009). In principle, pairing to the central region of the miRNA could also supplement pairingto the other regions of the miRNA, but a role for such pairing has been demonstrated only forsites that mediate Ago-catalyzed cleavage and not for sites that mediate destabilization andtranslation repression.

Here we describe “centered sites”, a unique class of microRNA target sites that lacks bothperfect seed pairing and 3’-compensatory pairing and instead has 11–12 contiguous Watson–Crick pairs to miRNA nucleotides 4–15. In the process of characterizing these sites, we foundthat Mg2+ concentration profoundly influences both the specificity and efficacy of miRNA-directed cleavage, and we performed whole-transcriptome analyses that substantially add tothe number of known instances in which metazoan miRNAs direct mRNA cleavage.

RESULTSA Unique Class of MicroRNA Target Site

The most highly conserved region of metazoan miRNAs is the 5’ region containing the seed(Lewis et al., 2003; Lim et al., 2003), which is the region most important for recognizing mosttargets. The next most highly conserved region spans nucleotides 13–16, which is the regionmost important for 3’-supplementary and 3’-compensatory pairing (Grimson et al., 2007).Despite being less conserved than other miRNA regions, we noted that the central region ofvertebrate miRNAs is significantly more conserved than is the opposite arm of the pre-miRNAhairpin (Figure 1A and S1A). Because both arms participate equally in the pairing required toform the pre-miRNA hairpin, preferential conservation of the miRNA observed in this regionsuggested that these central nucleotides play a role beyond that of miRNA biogenesis. One

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such role would be to aid in target recognition, but among the previously characterized targetingmodes, the central region is known to function only for cleavage sites, which seemed too rareto provide the additional selective pressure for conserving nucleotide identity at the miRNAcentral regions. Therefore, we searched for another type of site that might explain thispreferential conservation.

Examination of array data investigating the response of mRNAs after transfecting 11 miRNAsinto HeLa cells (Lim et al., 2005; Grimson et al., 2007) revealed a unique type of site that wasassociated with mRNA down-regulation (Figure S1B). This site type, which we call the“centered site”, was characterized by at least 11 nucleotides of contiguous Watson–Crick basepairing to the center region of the miRNA at either nucleotides 4–14 or 5–15, withoutsubstantial pairing to either the 5’ or the 3’ ends of the miRNA. Because of the location andextent of their base pairing, these sites occupy a unique position intermediate between seedsites and the extensively complementary cleavage sites (Figure 1B).

Because these sites are relatively rare, pooling of data from multiple miRNA transfections wasinitially required to achieve statistical significance for determining their efficacy. In order tosystematically analyze these sites, we therefore compiled additional array data from HeLaexperiments with similarly transfected miRNAs and siRNAs (Birmingham et al., 2006; Jacksonet al., 2006a; Jackson et al., 2006b; Schwarz et al., 2006; Anderson et al., 2008). To ensurethat the transfected mi/siRNAs were loaded and active within the silencing complex, the pooleddatasets were restricted to those of the 78 HeLa experiments for which the canonical 8mer3’UTR site to the transfected mi/siRNA was associated with downregulated mRNAs with highstatistical significance (P < 0.0001, K–S test, Table S1A). Testing matches that did not includethe canonical seed match to miRNA positions 2–7 showed that perfect 11-mer matches startingat miRNA positions 3, 4, and 5 were each significantly associated with repression (Figure 1C),whereas perfect 10-mer matches, perfect 9-mer matches, and near-perfect 11-mer matches(those with single mismatches or wobbles) were not significantly associated with repression(Figure S1C and D).

The efficacy of centered sites matching ectopically introduced miRNAs and siRNAs raised thepossibility that such sites might also mediate endogenous miRNA targeting. Array resultsexamining the effects of miRNA loss in zebrafish embryos lacking Dicer provided data on asufficient number of messages with centered sites to enable a systematic analysis of targetinginteractions in vivo. MicroRNAs present at 24 hours post fertilization (hpf), the developmentalstage used for mRNA analysis, were identified by high-throughput sequencing (Table S1B).Sites were considered for 21 of these miRNAs for which the canonical 8mer 3’UTR sites weresignificantly associated with mRNAs derepressed in the dicer mutant (P < 0.01, Table S1B).As observed for the ectopic interactions, perfect 11-mer matches starting at miRNA positions3, 4, and 5 were each associated with significant repression, although efficacy of sites startingat position 3 was mostly attributable to overlap with the “shifted 6mer” seed match (Figure1D), which comprises pairing to nucleotides 3–8 (Friedman et al., 2009). Perfect 10-mermatches, perfect 9-mer matches, and near-perfect 11-mer matches generally were notassociated with significant repression, although for a few of the numerous possibilitiesexamined, marginal significance was observed (Figure S1E and F).

When considering both ectopic and endogenous interactions, contiguous Watson–Crick3’UTR pairing to the central region of the miRNA, at either nucleotides 4–14 or 5–15, wasunique among the tested possibilities in that it was both consistently associated with mRNArepression and not attributable to overlap with previously described site types. A previous arraystudy had reported a handful of siRNA off-targets with similarly long stretches of contiguousWatson–Crick base pairing, but these sites were offset further towards the 3’ end of the miRNA,at nucleotides 6–16 or 7–17 (Jackson et al., 2003), a region not significantly associated with

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targeting when examined using our larger datasets. Effective miRNA-target predictionalgorithms rely heavily on perfect pairing to the seed region, and thus miss this additional classof targets (Bartel, 2009).

The transfected mi/siRNAs had an average of 11 and a median of eight centered sites in 3’UTRsof human mRNAs. About a quarter of the mRNAs with a centered site lacked conventionalseed sites to the transfected RNA and were sufficiently expressed in HeLa such that changescould be accurately measured on the arrays. Analysis of cumulative distributions of log-foldchanges indicated that >20% of these mRNAs responded to the transfected mi/siRNAs in amanner attributable to the site, with a lower bound for site efficacy resembling that of canonical7-mer sites (Figure 1E). Likewise, >30% of the endogenous centered sites analyzed appearedto mediate repression in zebrafish embryos (Figure 1F).

To examine whether centered sites also function in other animals, we analyzed mRNA arraydatasets monitoring the impact of knocking down proteins required for Drosophila miRNAbiogenesis (Kadener et al., 2009). Following either Drosha or Dicer1 knockdown inDrosophila S2 cells, messages with 3’UTR centered sites matching the endogenous miRNAshad a significant propensity to be derepressed (Figure S1G and H, P = 0.00045 and 0.027,respectively, for Drosha and Dicer1 knockdown datasets, Table S1C and D).

To confirm that centered sites can be directly targeted by miRNAs, luciferase reporterconstructs and their mutant counterparts with disrupted pairing were prepared and tested inboth HeLa cells and S2 cells (Figure 1G and S1I). For three of the four UTR fragments tested,the sites reduced protein output in a manner that depended on the presence of both the wild-type site and the cognate miRNA. Taken together, the reporter and microarray results suggestedthat the centered site is a miRNA target site capable of downregulation comparable with thatobserved for single 7-nt seed sites. Although they are much less abundant than both seed-matched sites and sites with 3’-supplementary pairing, centered sites are present in similarnumbers as 3’-compensatory sites and could help explain the preferential conservationobserved in the central region of most miRNAs.

MicroRNA-directed Cleavage Detected at Centered SitesBecause of pairing to the central region of the miRNA, centered sites might be subject to AGO2-dependent cleavage similar to that occurring for known cleavage sites of plants and animals,which are more extensively paired (Yekta et al., 2004; Davis et al., 2005; Jones-Rhoades etal., 2006). To test this possibility, we employed an in vitro cleavage assay using S100 extractprepared from HeLa cells (Martinez and Tuschl, 2004; Shin, 2008), focusing on mRNAfragments containing centered sites for miR-21 or let-7g miRNA, which are abundant in HeLacells (Figure 2A, Table S4A). Cleavage was observed at the position expected for AGO2-catalyzed cleavage of the centered sites (Figure 2B).

To examine whether cleavage was also occurring in the cells, we tested for miR-21-directedcleavage of GSTM3 mRNA (moderately expressed in HeLa cells), using RNA ligase–mediatedrapid amplification of cDNA 5’ ends (5’-RACE). By directly cloning and sequencing the 5’end of the 3’ cleavage product, this assay can be used to validate miRNA-directed cleavage(Llave et al., 2002; Yekta et al., 2004). To increase the sensitivity of the assay, XRN1, the5’→3’ exonuclease responsible for degrading the 3’ cleavage product (Souret et al., 2004;Orban and Izaurralde, 2005), was knocked down (Aleman et al., 2007). 5’-RACE fragmentswithin ~50 bp of the expected cleavage site were cloned for sequencing. The 5’ ends for sevenof nine sequenced clones precisely matched that expected for cleavage at the centered site inthe cell (Figure 2C). These results indicated that for an endogenous mRNA targeted at acentered site by an endogenous miRNA, at least some transcripts underwent AGO2-catalyzedcleavage in the cell.

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Pairing Requirements for Cleavage are Sensitive to Mg2+ ConcentrationTo understand the specificity of cleavage at centered sites, miR-21 recognition of the K89mRNA fragment (Figure 2) was examined further. The K89 RNA sequence, which wasperfectly complementary to positions 5–16 of miR-21, was systematically mutated at eachnucleotide corresponding to miR-21 positions 1–16, substituting an A:C mismatch or a G:Uwobble for each Watson–Crick match, and substituting a Watson–Crick match for each of thetwo mismatches (Figure 3A). When using 5.8 mM Mg2+, as in Figure 2B, or 2.2 mM Mg2+,both of which were within the ~2–6 mM range used previously to study in vitro cleavage(Martinez and Tuschl, 2004;Gregory et al., 2005;Maniataki and Mourelatos, 2005;Miyoshi etal., 2005;Rand et al., 2005;Ameres et al., 2007;Wang et al., 2009a;Wang et al., 2009b),cleavage was retained after changing positions outside of the centered site and was reducedafter changing most positions within the centered site, although wobble pairs were tolerated atpositions 6 and 8 (Figure 3B, top two panels).

Mg2+ is essential for the in vitro cleavage reaction (Schwarz et al., 2004) but also has a strikingeffect on the relative stabilities of matched and mismatched RNA duplexes (Serra et al.,2002). Indeed, lowering the Mg2+ concentration increases the fidelity of RNA 2’-O-methylation, another reaction specified by Watson-Crick pairing between small guide RNAsand their targets (Appel and Maxwell, 2007). We found that lowering Mg2+ gave maximaltarget RNA cleavage specificity and efficacy for substrates that were extensively paired tomiR-21, whereas higher Mg2+ was optimal for more weakly pairing substrates (Figure 3B).For example, the cleavage of K89-21as RNA, which is fully paired to the miRNA, was themost efficient at 0.3 mM Mg2+, whereas cleavage of the wild-type K89 substrate containingthe centered site with only 12 contiguous pairs was undetectable at 0.3 mM Mg2+ and mostefficient at 5.8 mM Mg2+, and K89-m4GC, which had an intermediate number of contiguouspairs, had an intermediate Mg2+ optimum (Figure 3B and C).

The free-Mg2+ levels in the cytoplasm of various cells and tissues is less than 1 mM (Gunther,2006), a concentration at which we found that efficient cleavage required pairing moreextensive than that of typical centered sites (Figure 3B). Nonetheless, some cleavage at thecentered site was detected at physiological Mg2+ concentrations (Figure 3B, 0.75 mM Mg2+),which explained why the 5’-RACE assay yielded fragments diagnostic of miR-21-directedcleavage in the cell (Figure 2C).

Whole-transcriptome Analysis of miRNA-directed CleavageThe poor efficacy of cleavage at the centered site at physiological Mg2+ concentration calledinto question whether miRNA-directed cleavage plays a consequential role during repressionmediated by centered sites and suggested that most repression at centered sites might resemblethe destabilization and translational repression observed for most seed-matched targets. Tobetter characterize the scope of miRNA and siRNA-directed cleavage in mammals, and toexamine the extent to which cleavage at centered sites is relevant to target gene regulation invivo, we applied degradome sequencing to mammalian cells. Degradome sequencing generatesshort sequence tags representing the 5’ ends of uncapped mRNA fragments found in the cell(Addo-Quaye et al., 2008; German et al., 2008). Although these fragments are predominantly5’→3’ exonuclease degradation intermediates, they also include 3’ fragments of Argonaute-catalyzed mRNA cleavage in sufficient numbers to enable empirical detection of endogenouscleavage targets of plant miRNAs and siRNAs (Addo-Quaye et al., 2008; German et al.,2008). Inspired by this success in plants and the ability to detect miR-21-directed cleavage by5’-RACE, we applied the method to HeLa cells following XRN1 knockdown by RNAi (FigureS3A).

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Sequencing yielded 14,323,668 tags mapping to the human genome, with a diversity of2,069,190 unique tag sequences. Of the total tags, 61.2% came from protein-coding genes andrepresented 36,806 out of 46,319 ENSEMBL mRNAs (Figure 4A). The tags showed arelatively uniform distribution across the mRNAs, with a very strong peak at the 5’ terminus(Figure 4B). About 30% of tags were not classified because they did not map to matureannotated RNAs (Figure 4A). Many of these were from introns and processing fragments frompri-miRNAs, mitochondrial tRNAs, ribosomal RNAs, and snRNAs, illustrating how unstable3’ products of endonucleases can be detected in mammalian cells by using degradomesequencing (Table S2A and B).

To determine if miRNA centered sites were associated with cleavage at the expected positionwithin the mRNA 3’UTR, we searched for centered matches to 50 distinct, conserved miRNAsmost highly expressed in HeLa cells and tabulated the frequency of degradome tagscorresponding to mRNA cleavage at the tenth position of these sites. Tags corresponding tocleavage at the expected position were found much more frequently for authentic miRNA:sitepairs than for negative-control pairs (Figure 4C). However, when we excluded miR-196a,miR-151, and miR-28, which target several extensively paired sites, the signal abovebackground was greatly reduced, suggesting that most centered sites lacked thecomplementarity required for robust miRNA-directed cleavage (Figure 4C). The abundanceof degradome tags mapping to the expected cleavage sites of the siRNAs targeting XRN1illustrated that the method can identify tags diagnostic of AGO2-catalyzed cleavage in humancells (Figure S3). These results supported those from the in vitro cleavage assays (Figure 3B)in suggesting that under physiological Mg2+ conditions the mRNA downregulation mediatedby centered sites is usually accompanied by very little AGO2-catalzyed cleavage.

Genome-wide Search for miRNA:site Duplexes with High ComplementarityOur observation of significant cleavage at the small subset of centered sites with unusuallyextensive complementarity to the miRNA indicated that miRNA-directed cleavage atextensively paired sites was more frequent in animals than had been appreciated. This insightprompted a systematic examination of mammalian sites with extensive miRNAcomplementarity of the type that would mediate cleavage in plants, but might not have fulfilledour criteria for classification as centered sites because they either had perfect seed pairing orlacked 11 contiguous pairs within positions 4–15.

To search for potential cleavage sites in mammals, we used a scoring rubric similar to thosethat successfully identify miRNA target sites in plants (Figure 5A) (Jones-Rhoades and Bartel,2004; Allen et al., 2005). The search yielded 106 predicted miRNA:site duplexes scoring ≤2.0(Figure 5B), including 47 in annotated ORFs, 16 in 5’UTRs, and 43 in 3’UTRs (Table S3A).At the mid-to-higher penalty scores, sites were no more abundant than expected by chance,but at scores ≤3.0, sites were at least 1.5-fold enriched compared to the control sets of chimericmiRNAs constructed so as to preserve the seeds as well as the overall dinucleotide andtrinucleotide compositions of authentic miRNAs (Figure 5C). Repeating the analyses withannotated murine miRNAs yielded analogous results (Figure S4C–E, Table S3B).

The higher abundance of extensive matches to miRNAs compared to that of controls mightindicate biological function. However, eukaryotic genomes, complex tapestries containingremnants of innumerable duplications and repetitive elements, are far from random, and thusthis abundance might simply be a consequence of the miRNAs and sites sharing commonancestry. To distinguish between these possibilities, we examined the conservation oforthologous sites in five mammalian species, as assessed using a conservation-alignment (CA)score (Figure 5D). When applied to sites for distinct miRNAs conserved throughout mammals,17 miRNA:site duplexes had CA scores ≤3.0 (Figure 5E), most of which were unlikely to be

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conserved by chance (Figure 5F). Four of the 17 top-scoring sites were miR-151-5p targets(Table S3C).

Cleavage at Highly Complementary Predicted DuplexesHaving found evidence that the most extensively paired sites were more abundant and moreconserved than expected by chance, we returned to the degradome sequencing data to searchfor evidence that these sites were cleaved in the cell. Because the degradome sequencing dataincluded intermediates of normal mRNA decay, steps were taken to distinguish AGO2cleavage products from other decay intermediates. To do this, we considered the tag possessionratio (TPR), which represented the proportion of predicted miRNA:site duplexes that wererepresented by tags at the expected cleavage site (Figure 6A). When focusing on the miRNAsand mRNAs expressed in HeLa, miRNA:site duplexes with alignment penalty scores ≤2.5possessed significantly more cleavage tags at the expected cleavage site than did controlduplexes (Figure 6B and Table S4B, Fisher’s exact test, P = 1.1 × 10 −04). Even after excludingtags mapping to multiple loci, this TPR difference remained both substantial and significant(Figure 6C and Table S4B, P = 2.6 × 10 −04). MicroRNA-directed cleavage in Arabidopsissometimes occurs at ±1 nt from the expected cleavage site (Addo-Quaye et al., 2008). Whenapplying a window of ±1 nt, there was no improvement in the TPR of expressed miRNA:sitepairs (Figure S5A and Table S4B). As an added control, we repeated the analysis for miRNAsthat were not expressed in HeLa cells and found that these miRNAs performed similarly to thechimeric miRNA controls (P = 1.0) and significantly worse than the miRNAs expressed inHeLa cells (P = 5.3 × 10 −05). These results strongly indicated that for miRNA:site pairs withfavorable alignment scores (≤2.5), most tags at the expected cleavage site did not arise frombackground 5’→3’ degradation but instead were the consequence of miRNA-directed mRNAcleavage.

MicroRNA-directed Cleavage in HeLa Cells and Human brainUsing an alignment penalty score of 2.5, a threshold at which the cumulative TPR differencebetween signal and background was most significant in HeLa data (Table S4B), we found eightmiRNA-directed cleavage targets with tags precisely at the expected cleavage site (Table 1and Figure S5B). All eight cleavage sites were in 3’UTRs, and half were conserved in othermammals (Table 1 and Figure S5B). Four of the pairs involved miR-151-5p (Figure 6E–G andTable 1). miR-196a and its cleavage target HOXB8 are both known to be moderately expressedin HeLa cells (Lim et al., 2005), and as expected HOXB8 was among the eight (Figure 6H).

To extend our results beyond cells in culture, we performed degradome sequencing using poly(A)-selected RNA from whole human brain. Sequencing yielded 9,240,114 reads mapping tothe human genome, with a diversity of 2,360,502 unique tag sequences. MicroRNAs expressedin human brain tissues were found by small-RNA sequencing (Table S4C). As in HeLa cells,we found a statistical association between the miRNA:site pairs and cleavage tags for miRNAsand mRNAs expressed in brain (Figures 6D and S5D). For pairs with alignment score ≤3.0,the TPR was significantly higher than for that of the controls (Table S4B, P = 0.008 and P =0.030, nonexpressed and chimeric controls, respectively). Statistical significance was retainedwhen also including tags mapping 1 nt downstream of the expected cleavage site as diagnosticof cleavage (Table S4B, P = 0.011 and P = 0.013, nonexpressed and chimeric controls,respectively), perhaps because some 5’→3’ trimming occured in the animal, where we couldnot knock down XRN1 activity. Eleven sites with scores ≤3.0 had tags suggestive of miRNA-directed cleavage (Table S4D) at the expected position (Table 1) and two had tags suggestiveof cleavage at position −1 (Figure S5E). Three of the 13 matched miR-151-5p and includedN4BP1, which was also identified in HeLa cells. FRS2, a proposed target of miR-182, was alsoidentified in HeLa cells. Four of the miRNA:site pairs newly identified in brain appearedconserved in other mammals (CA ≤3.0; Table 1 and Figure S5E).

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DISCUSSIONCentered Sites

We present centered sites as a type of miRNA target site. Centered sites contain at least 11contiguous nucleotides that pair to a miRNA at positions 4–14 or 5–15, a pairing pattern distinctfrom that of most 3’-compensatory sites and seed sites. However, because a centered site mightinclude additional nucleotide pairing on either side and a 3’-compensatory site might haveadditional pairing extending into the miRNA central region, there is potential overlap betweena few extended centered sites and a few 3’-compensatory sites. Similarly, a seed site mightinclude 3’-supplementary pairing extending into the miRNA central region, which createspotential overlap between a few extended centered sites and a few 3’-supplementary sites.However, such overlap with previously known site types is very rare. For example, a searchof annotated human 3’UTRs revealed that for most human miRNAs, no seed-matched sitesextend into centered sites; i.e., most human miRNAs have no 3’UTR match with contiguousWatson–Crick pairing to nucleotides 2–14. Furthermore, conservation analysis and array datashow that seed-type targets prefer to acquire supplemental pairing at positions 13–16 ratherthan extending pairing through nucleotides 9–12 (Grimson et al., 2007).

The reason that centered sites had not been described previously can be explained by theirrelatively low abundance, which resembles that of 3’-compensatory sites and is far lower thanthat of seed-matched sites. Although no more effective than 7-nt seed-matched sites, centeredsites are 4 nt longer, leading to an informational complexity ~250-fold (~44-fold) greater thanthat of 7-nt sites and a correspondingly increased difficulty for their emergence and retentionduring evolution. The rarity of centered sites hampers statistical assessment of whether theyare subject to evolutionary conservation. Nonetheless, the conserved miRNAs of mammalseach match an average of 13 centered sites in human 3’UTRs (Figure S1J), and based on ourzebrafish analyses we estimate that on average about two sites per miRNA both reside inmessages coexpressed with the miRNA and mediate repression. The presence of even a fewbeneficial interactions (species-specific or more broadly conserved) for a subset of the miRNAscould impart at least intermittent pressure to preserve the miRNA sequence, thereby explainingthe preferential conservation observed in the central region of vertebrate miRNAs (Figure 1A).Moreover, centered sites resemble 3’-compensatory sites in providing a mechanism by whichdifferent members of the same miRNA seed family can repress distinct targets (Bartel, 2009).

Why would centered sites require so much more contiguous pairing than that required by seedsites? When bound by the Argonaute protein within the silencing complex, the seed is thoughtto be pre-organized to favor Watson-Crick pairing to the mRNA (Bartel, 2004). In the currentversion of this seed-nucleation model, pairing cannot propagate to the center of a miRNAwithout a substantial conformational change in which the original contacts between Argonauteand the miRNA central and 3’ regions are disrupted (Bartel, 2009). Disrupting these contactsoffsets some binding energy gained in forming the central pairs, causing contiguous pairingadjacent to the seed to contribute less affinity than might have otherwise been expected. Thislower contribution of pairing to the central region, combined with the higher contributionachieved by the pre-organized seed, would explain why so much more pairing is needed forcentered sites to achieve the same outcome as 7-nt seed sites.

Mg2+ Effect on Cleavage Specificity and EfficiencyOur results shed a new light on the biochemistry of RNAi. We suggest that at 37°C in the lowMg2+ concentrations present in the cell, only the extensively paired sites can be bound withthe stability and conformation that favors mRNA cleavage, and that after cleavage the productsare not so tightly bound so as to slow multiple turnover. In higher Mg2+, however, lessextensively paired sites achieve the stability and conformation needed for cleavage, and

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product release is more apt to slow turnover. This model explains the reduction of bothspecificity and efficiency at extensively paired sites observed in high Mg2+ concentrations.Under these conditions, less extensively paired sites are more readily cleaved—hence, thereduced specificity. The more extensively paired sites, on the other hand, undergo slowerproduct release and gain little benefit from this more permissive binding-and-cleavage regime.Indeed, any benefit gained is more than offset by the tighter binding of the miRNA to lessextensively paired sites, which causes the total cellular RNA present in extracts used forcleavage reactions to more effectively inhibit utilization of the labeled substrates—hence, thereduced efficiency. The free cytoplasmic Mg2+ concentration in most cells and tissues is < 1mM (Gunther, 2006), suggesting that cleavage specificity is very high in vivo.

Our results explain previous observations regarding the effects of adding phosphate-containingcompounds to in vitro cleavage reactions. Many diverse phosphate compounds, includinginorganic monophosphate, stimulate the multiple-turnover cleavage by the mammaliansilencing complex (Gregory et al., 2005). We suggest that these phosphate compounds titratethe free Mg2+, which in turn increases product turnover through decreased RNA duplexstability.

Endogenous miRNA-directed mRNA Cleavage in HumanWe find that miRNA-directed cleavage of mammalian mRNAs, although even more rare thanrepression at centered sites, occurs more frequently than previously appreciated. Twoendogenous cleavage targets had been reported in mammals, HOXB8 and RTL1 (Yekta et al.,2004)(Davis et al., 2005). We substantially add to this list, with evidence for cleavage of sevenadditional targets in HeLa cells and cleavage of thirteen in human brain, two of whichoverlapped with HeLa targets. This small overlap, largely attributed to differential expressionof the miRNAs or mRNAs in the two samples (Tables 1, S4A, S4C and S4D), suggests that asmore tissues are examined, more cleavage targets will be found.

The fraction of degradome sequencing tags that provided evidence of miRNA-directedcleavage was generally higher in the HeLa analysis than in the brain analysis (Table 1; FigureS5B and S5E). In the brain, this fraction of cleavage tags was sufficiently low so as to suggestthat some might represent degradation intermediates not indicative of miRNA-directedcleavage. Whether a smaller fraction of brain messages are cleaved, however, is unclear. Thebrain analysis lacked the benefit of the XRN1-endonuclease knockdown, designed to stabilizethe transient 3’ cleavage product so that it could be more readily detected over the backgroundof metastable mRNA-decay intermediates. Moreover, whole brain has many cell types, withthe possibility that differential expression of a miRNA and its cleavage targets might decreasethe signal relative to background. Nonetheless, for most cleavage targets in HeLa and for somein brain, degradome profiles resembled those of plant targets with validated biologicalrelevance (Figures 6F–H, S5Bi and S5Ei) (Addo-Quaye et al., 2008;German et al., 2008),strongly supporting the hypothesis that the miRNA-directed cleavage pathway is an importantdegradation pathway for those mRNAs.

In both brain and HeLa cells, several cleavage targets identified were targets of miR-151-5p.This miRNA derives from a hairpin that has homology to the L2 subclass of repeat elementsknown as long interspersed nuclear elements (LINEs). L2 LINE elements are remnants of anon-LTR retrotransposon activity present in the common ancestor of mammals. They makeup over 3% of the human genome (Kamal et al., 2006). Indeed, the miR-151 hairpin is derivedfrom a tail-to-tail arrangement of two L2 fragments (Figure 6E). Hence, miR-151-5p derivedfrom L2(+) is strongly complementary to several target sites derived from L2(−) repeats.Analogous tail-to-tail arrangements of short (S)INE fragments produce transcripts with longerhairpins that are processed in mouse ES cells into endogenous siRNAs (Babiarz et al., 2008).However, miR-151-5p and miR-151-3p are typical miRNAs, in that 1) their accumulation

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depends on both Drosha/DGCR8 and Dicer endonucleases (Babiarz et al., 2008), 2) they pairto each other with 2-nt 3’ overhangs, 3) they are the two dominant products accumulating fromthe hairpin (Figure S5C), and 4) their hairpin has a conservation pattern typical of otherconserved miRNAs (Figure 6E).

Two other miRNAs that direct cleavage in HeLa, miR-28-5p and miR-545*, are also L2 repeat–derived miRNAs. The notion that these miRNAs and their targets ultimately derived from thesame ancestral elements is reminiscent of the origin of some plant miRNAs, which derive fromduplicated fragments of their cleavage targets (Allen et al., 2004; Rajagopalan et al., 2006). Inmammals, however, the miRNAs and target sites evolved in parallel from the commonancestor, rather than one from the other. Moreover, in mammals, common ancestry betweenthe miRNAs and their targets can be detected for older, conserved miRNAs, such as miR-151and miR-28, whereas in plants common ancestry has been detected only for younger,nonconserved miRNAs.

The observation that many of the cleaved mRNAs were the targets of repeat-derived mRNAscan be explained by the fact that repeat-derived miRNAs are more likely to encounterextensively complementary matches, since repeat-element remnants are found within manymRNAs. Over the course of evolution, repeat-derived miRNAs presumably had access to awide variety of cleavage targets, providing the opportunity for some favorable regulatoryinteractions to emerge and be retained as conserved cleavage interactions. Thus, the repeat-derived miRNAs and their cleavage targets provide yet another avenue for repetitive elementsto shape the regulation of cellular genes.

Concluding RemarksThe discovery of centered sites raises the question of how many additional site types remainto be found. On the one hand, transcriptome/proteome changes observed after introducing ordeleting a miRNA can all be explained by direct interactions between the miRNA and messageswith the five known site types (seed sites, 3’ supplementary seed sites, 3’ compensatory sites,centered sites, and cleavage sites), combined with indirect effects as changes in the primarytargets influence expression of secondary targets. On the other hand, detailed experimentalfollow-up on mRNAs that respond to the miRNA despite lacking any of these established sitetypes seems to indicate that some of them should not be dismissed as secondary targets butmight instead be direct targets (Lal et al., 2009). However, the pairing schemes proposed thusfar for these unusual interactions have not been defined sufficiently to provide predictive utility.That is, in contrast to centered sites and the previously known site types, these pairing schemeslack the specificity required to distinguish other responsive messages with similar pairing frombackground. Hence, experiments like that shown in Figure 1C–F cannot distinguish responsivemessages that satisfy these unusual pairing schemes from nonresponsive messages that do not.Perhaps unknown factors binding to neighboring UTR elements help achieve interactionspecificity differently for each individual mRNA in a manner too idiosyncratic to begeneralized into site types. Alternatively, future insights into miRNA targeting might identifycommonalities in these unusual interactions, which could form the basis of novel site typeswith predictive value.

EXPERIMENTAL PROCEDURESA detailed description of all materials and methods used can be found in the SupplementalText.

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Microarray and Molecular AnalysesArray analyses were as in (Grimson et al., 2007). Luciferase reporter constructs were preparedas in (Grimson et al., 2007), and assays were performed as in (Farh et al., 2005). In vitrocleavage reactions were essentially as in (Haley and Zamore, 2004; Shin, 2008). Uncapped 5’ends of GSTM3 mRNA degradation products were identified using the 5’-RACE kit(Invitrogen), as in (Jones-Rhoades and Bartel, 2004), starting with cells in which XRN-1 mRNAwas knocked-down more than 90%, as confirmed by RT-PCR (Aleman et al., 2007).Degradome libraries were constructed essentially as in (Addo-Quaye et al., 2008). Small-RNAlibraries were prepared for Illumina sequencing as described (Grimson et al., 2008).

Analysis of miRNA ConservationOut of 223 miRNA genomic loci producing 197 mouse miRNAs conserved in other mammals(Friedman et al., 2009), 203 miRNA loci producing miRNAs with 5’ ends validated from alarge scale profiling of mouse miRNAs (Chiang et al. 2010) were used in the analysis of Figure1A.

Processing of Degradome TagsAfter removing linker sequences and tags shorter than 20 nt, degradome tags were mapped toRNAs annotated in the ENSEMBL (http://www.ensembl.org/), requiring a perfect match. Tofind “multiple loci tags”, and tags that did not map to annotated RNAs, filtered tags weremapped to the human genome (hg18, http://genome.ucsc.edu/). When determining TPRs,filtered tags were mapped to a curated set of distinct mRNAs (Baek et al., 2008). ExpressedmRNAs were those represented by at least one degradome tag.

miRNA:site DuplexesWhen searching for miRNA:site duplexes, distinct mRNAs and miRNAs were selected toavoid over-counting predicted duplexes involving miRNA families or mRNA isoforms. Toselect distinct miRNAs, all human miRNAs and miRNA* sequences (miRBase 11.0) werealigned and classified into groups whose members differed from each other at ≤5 positions.The miRNA with the lowest miRBase annotation number was selected as the representativefrom each group. For distinct mRNAs, the mRNA isoform with the longest 3’UTR (or, if all3’UTRs were of the same length, a randomly chosen isoform) was selected from a previouslyfiltered set of RefFlat and H-INV annotations (Baek et al., 2008).

CA ScoreTo search for orthologous sites, we used 165 distinct miRNAs conserved among mammals anda 6-way genome alignment (human, mouse, rat, dog, horse, and pig) from the UCSC genomebrowser (hg18, http://genome.ucsc.edu/). Alignment penalty scores were determined and thesecond worst score rather than the worst score was selected as the CA score to accommodatesome genome-alignment errors, incomplete genome sequences, and species-specific losses.

Generation of miRNA-like Control SequencesTo generate controls with the same seed composition and same trinucleotide composition asauthentic miRNAs, chimeric miRNA sequences were created by reciprocally recombining,using the link between nucleotides 10 and 11 as the crossover breakpoint, two miRNAsrandomly chosen (without replacement) from miRNA pairs with the same dinucletide atpositions 10 and 11 considering only our set of distinct miRNAs. Ten chimeric miRNA cohortswere generated to estimate the signal-to-background ratios.

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http://www.ensembl.org/

http://genome.ucsc.edu/

http://genome.ucsc.edu/

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank Andrew Grimson, Daehyun Baek, and Alexander Subtelny for helpful discussions, Shujun Luo and GarySchroth for Illumina sequencing of the small-RNA library from brain, and the Whitehead Genome Technology Corefor the remaining Illumina sequencing. Supported by a Damon Runyon postdoctoral fellowship (C.S.) and a grantfrom the N.I.H. D.B. is a Howard Hughes Medical Institute Investigator.

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Figure 1. Centered Sites Regulate both mRNA Accumulation and Protein Output(A) Conservation of 4-nt segments of mammalian miRNAs. As schematized in Figure S1A,segments from the mature miRNA (orange) were compared with opposing segments from theother arm of the hairpin (grey). At each position of the mouse miRNAs, the number of segmentsperfectly conserved in the whole-genome alignments of the other 29 species (Blanchette et al.,2004) is plotted.(B) Spectrum of functional miRNA target sites.(C) Response of HeLa mRNAs with contiguous perfect pairing to 11-mer sites starting at theindicated positions of 78 transfected mi/siRNAs. Plotted are mean fold changes for mRNAswith 3’UTR sites to the cognate mi/siRNAs (gray) and the cohorts of chimeric miRNA-like

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controls sequences (white, error bars indicate standard deviation for 10 cohorts). Also plottedis the mean expression change for mRNAs with a shifted 6-mer 3’UTR site (purple), whichincludes all mRNAs with 11-mer sites starting at position 3. The 11-mer sites with repressiondistributions significantly different from that the no-site distribution are indicated (*, P < 0.05;** P < 0.01; K–S test).(D) In vivo response of mRNAs with 11-mer sites to endogenous miRNAs after loss of allmiRNAs. Plotted is the fold change of zebrafish mRNAs with 11-mer sites to 21 endogenousmiRNAs depleted in the dicer mutant. Otherwise as in panel (C).(E) Reduced levels of HeLa messages with either centered sites or canonical sites to 78transfected mi/siRNAs. Shown is analysis of microarray data, plotting cumulative changes ofmRNAs with single 3’UTR sites of the indicated types. Canonical sites (8mer, 7m8, 7A1, and6mer) had either 8, 7 or 6 nt matches centered on the miRNA seed (Bartel, 2009); centeredsites had 11 contiguous Watson–Crick pairs to miRNA positions 4–14 or 5–15; control siteswere centered sites to the chimeric miRNA-like control sequences (combining results for all10 cohorts).(F) Efficacy of endogenous centered sites in vivo. Shown is analysis of microarray data, plottingcumulative changes of zebrafish mRNAs with single 3’UTR sites to 21 miRNAs depleted inthe dicer mutant. Otherwise as in panel (E).(G) miRNA-mediated repression at centered sites. Shown is the fold repression of luciferasereporter genes fused to 3’UTR fragments of the indiciated genes with the indicated sites ormutant sites. Plotted are the geometric means, normalized to the geometric means observedfor reporters with mutant sites. Error bars represent the second largest and the second smallestvalues among 12 replicates (from 4 independent experiments). Statistical significance isindicted (**, P value < 0.001, Wilcoxon rank-sum test). See also Figure S1 and Table S1.

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Figure 2. MicroRNA-directed Cleavage at Centered Sites(A) Centered sites for miR-21 or let-7g within 3’UTR fragments of the indicated mRNAs testedin cleavage assays. K89, KIAA1189; FL9, FLJ40919; NFI, NFIA (sequences provided inSupplementary Information).(B) Cleavage of target fragments directed by endogenous miRNAs in vitro. 5’-Cap-radiolabeled mRNA fragments were incubated in HeLa cytoplasmic S100 extract for theindicated time and analyzed on denaturing gels. As a control, fragments modified to be fullycomplementary to the cognate miRNA, designated as antisense (as) substrates, were tested andanalyzed in parallel. Whereas most fragments were cleaved predominantly at the expected site,NFIA was cleaved at two positions (*, Figure S2), as is sometimes observed in vitro (Martinezand Tuschl, 2004).(C) miR-21–directed cleavage of GSTM3 mRNA in HeLa cells. 5’-RACE using primersspecific for GSTM3 mRNA was performed on mRNA isolated from HeLa cells treated withtwo siRNAs targeting XRN-1. Seven of nine sequenced clones mapped to the position expectedfor miR-21–directed cleavage. The other two clones mapped 52 nt downstream.

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Figure 3. Pairing Requirements for Cleavage at a Centered Site and the Influence of Mg2+

Concentration(A) Sequences used to examine pairing requirements for cleavage. Seqeunces were derivativesof the K89 3’UTR fragment, a miR-21 target. K89-21as, fully complementary version of K89;K89-m1, matched to position 1 of miR-21; k89-mm2, A-C mismatch at position 2; K89-mm3GU, G:U wobble pairing position 3.(B) The influence of Mg2+ on cleavage specificity and efficacy in vitro. Reactions wereperformed as in Figure 2B, using the substrates depicted in (A), with the indicatedMg2+concentrations. Quantification of the fraction cleaved is plotted on the right.(C) Plot of the effect of Mg2+ on cleavage efficacy for the model centered site (K89), a moreextensively paired site (K89-m4GC), and a fully paired site (K89-21as).

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Figure 4. Rare MicroRNA-directed Cleavage Detected by Degradome Sequencing(A) Mapping of HeLa degradome sequencing tags to the transcriptome. Antisense correspondsto tags mapping antisense to annotated mRNAs, noncoding RNAs (ncRNAs), pseudogenes,and mitochondrial (MT) RNAs. Coverage indicates the fraction of the 46,319 annotatedmRNAs that were represented by at least one tag. Unclassified tags mapped primarily to intronsand 3’ flanking regions of ncRNAs (Table S2A).(B) The distribution of degradome tags along the length of the mature mRNAs. mRNAs weresplit along their length into 100 bins, and tag 5’ ends were tallied for each bin. Shown are theaggregate tallies for all mRNAs.

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(C) Search for evidence of cleavage at centered sites. Plotted are the numbers of interactionswith evidence for cleavage at the expected position (0) or at positions either upstream (negativevalues) or downstream (positive values). Interactions were counted if a centered site matchinga conserved miRNA expressed in HeLa had a tag supporting cleavage at the indicated position(blue). Analysis excluding miR-196, miR-28, miR-151-5p is also shown (red). As a control,the analysis was repeated using 10 cohorts of artificial tags, generated by randomly positioningtags on mRNAs (gray; error bars, standard deviation). See also Figure S3 and Table S2.

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Figure 5. Enrichment and Conservation of miRNA:site Duplexes with Extensive Complementarity(A) Illustration of the alignment penalty score, used to judge the quality of pairing to miRNAs.Pairing to miRNA nucleotides 2–21 was considered, assigning a 2-point penalty for eachmismatch or alignment insertion/deletion (indel) within the miRNA core (nucleotides 2–13)(Mallory et al., 2004), a 1-point penalty for each mismatch or insertion/deletion outside thecore, or each G:U wobble within the core, a 0.5-point penalty for each G:U wobble outside thecore. An additional 1-point penalty was assigned to sites lacking an A across from miRNAnucleotide 1 (Lewis et al., 2005).(B) Distribution of scores for potential miRNA:site duplexes with at least seven consecutivebase pairs and 13 base pairs in total. Sites were considered for the 620 distinct human miRNA/miRNA*s annotated in miRBase, version 11.0, excluding four miRNAs that paired to multiplerepeat loci, skewing the distribution to the left (Figure S4B).(C) Analysis of site enrichment. To estimate the signal-to-background ratio for each score bin,the number of miRNA:site duplexes was compared with the number of miRNA:site duplexesfound when using chimeric control miRNAs (error bars, standard deviation for 10 chimericmiRNA cohort sets).(D) Illustration of the conservation alignment (CA) score, used to identify conservedmiRNA:site pairs. Alignment penalty scores were considered for human sites aligned inorthologous genomic regions of mouse, rat, dog, horse, and pig, with the second highest (secondworst) among the six assigned as the CA score.(E) Distribution of CA scores for miRNA:site duplexes. Sites were considered for 165 distinctmiRNAs conserved in mammals.

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(F) Analysis of preferential conservation of extensively paired sites. To estimate the signal-to-background ratio for each score bin, the fraction of miRNA:site duplexes that were conservedwas compared with the fraction of analogous duplexes that were conserved when usingchimeric control miRNAs, accounting for the lower abundance of matches to control sequences(error bars, standard deviation for 10 chimeric miRNA cohort sets). See also Figure S4 andTable S3.

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Figure 6. Enrichment of Degradome Tags at Sites of Expected miRNA-directed mRNA Cleavage(A) The tag possession ratio (TPR), used to search for evidence of miRNA-directed cleavage.At each alignment penalty score, the number of miRNA:site duplexes that had at least one tagwith its 5’ terminal nucleotide mapping to the expected site of cleavage (across from miRNAposition 10) was tallied, as was the number duplexes that lacked a tag indicative of cleavage.The TPR was calculated as the number of duplexes with a tag divided by the sum of all duplexes.(B) Enrichment of tags at the expected sites. TPR values for miRNAs expressed in HeLa (blue;Table S4A) were compared to values for miRNAs not expressed in HeLa (red) and values for10 cohorts of chimeric control miRNAs (gray; error bar, the standard deviation).(C) Enrichment of tags at expected site after excluding tags mapping to multiple loci.(D) Enrichment of tags at the expected sites, after omitting tags mapping to multiple loci, inhuman brain. TPR values for miRNAs expressed in human brain (blue line; Table S4C) werecompared to values for miRNAs not expressed in human brain (red) and values for 10 cohortsof chimeric control miRNAs (gray; error bar, the standard deviation).(E) The mir-151 hairpin and its cleavage targets. Schematic depicts the mir-151 hairpin, thepositions of the two ancestral L2 LINE repeats that gave rise to the hairpin, the region of highconservation in sequenced mammals. Once processed from the hairpin the mature miR-151-5ppairs to and directs cleavage of mRNAs in HeLa cells.(F–H) The distribution of degradome tags along the length of the indicated mRNAs (omittingtags mapping to >10 genomic loci). The red peaks are cleavage tags corresponding to cleavageat the site expected for the indicated miRNA. Shown are results from HeLa cells. Similar graphsare provided for the other mRNAs with evidence of miRNA-directed cleavage in HeLa cells(Figure S5B) and in human brain (Figure S5E). See also Figure S5 and Table S4.

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alty

scor

es (S

core

) for

whi

ch th

e TP

R (c

ount

ing

only

tags

map

ping

pre

cise

ly to

the

expe

cted

cle

avag

e si

te)

sign

ifica

ntly

exc

eede

d ba

ckgr

ound

(≤2.

5 in

Hel

a ce

lls a

nd ≤

3.0

in b

rain

; Tab

le S

4B).

The

expr

essi

on o

f eac

h m

iRN

A is

indi

cate

d by

its m

iRN

A re

ads i

n a

high

-thro

ughp

ut se

quen

cing

exp

erim

ent (

Tabl

e S4

A a

nd S

4C).

Cle

avag

e ta

gs w

ere

thos

e ta

gs m

appi

ng p

reci

sely

to th

e ex

pect

ed si

te o

f cle

avag

e an

d w

ere

norm

aliz

ed b

y th

e nu

mbe

r of t

imes

they

map

ped

to th

e ge

nom

e. F

or e

ach

mR

NA

the

frac

tion

of d

egra

dom

e ta

gs th

at w

ere

clea

vage

tags

is in

dica

ted

(Deg

rado

me

frac

tion)

. Site

s with

CA

≤3.

0 ar

e ca

tego

rized

as c

onse

rved

(Fig

ure

S5B

and

S5E

)

Tis

sue

miR

NA

miR

NA

rea

dsm

RN

AL

ocat

ion

of si

teSc

ore

Cle

avag

e ta

gsD

egra

dom

e fr

actio

n (%

)C

onse

rved

HeL

a ce

llm

iR-1

96a

3682

HO

XB8

3’U

TR1

150

.0*

miR

-545

*34

IGFB

P43’

UTR

, LIN

E L2

21

0.7

miR

-28-

5p18

29LY

PD3

3’U

TR, L

INE

L22.

50.

753.

4

miR

-151

-5p

2526

N4B

P13’

UTR

, LIN

E L2

255

14.1

Yes

miR

-151

-5p

2526

MPL

3’U

TR, L

INE

L21.

50.

56.

9

miR

-151

-5p

2526

LYPD

33’

UTR

, LIN

E L2

04

17.9

miR

-151

-5p

2526

ATPA

F13’

UTR

, LIN

E L2

11

1.4

Yes

miR

-182

624

FRS2

3’U

TR2.

51

0.7

Yes

Hum

an b

rain

miR

-28-

5p25

44M

DG

A13’

UTR

, LIN

E L2

31

0.1

miR

-151

-5p

33,0

07M

DG

A13’

UTR

, LIN

E L2

39

0.9

miR

-151

-5p

33.0

07N

4BP1

3’U

TR, L

INE

L22

62.

1Y

es

miR

-873

2033

MAN

2C1

OR

F2.

51

0.2

Yes

miR

-330

-5p

744

FAM

62C

OR

F3

21.

3

miR

-95

523

EGLN

33’

UTR

2.5

11.

2

miR

-182

115

FRS2

3’U

TR2.

52

1.4

Yes

miR

-877

41SP

TBN

1O

RF

32

0.1

miR

-185

1014

PMVK

5’U

TR3

20.

8

miR

-383

593

DC

TN4

OR

F2

10.

4Y

es

miR

-598

8131

EFTU

D2

OR

F2.

51

0.3

* Alth

ough

miR

-196

a:H

OXB

8 w

as n

ot c

lass

ified

as c

onse

rved

usi

ng o

ur C

A sc

ore

beca

use

the

site

is m

issi

ng in

pig

and

hor

se, t

his p

airin

g is

kno

wn

to b

e co

nser

ved

in m

ore

dist

ant l

inea

ges,

incl

udin

g fr

og a

ndfis

h (Y

ekta

et a

l., 2

004)

.


author manuscript nih public access jin-wu nam kyle kai-how … · chanseok shin1,2,3,4,6, jin-wu...

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